Dyes are compounds used to add color or fluorescence to any kind of material or matrix, e.g. textiles, paper, plastics, biological molecules, cellular structures and so forth. To be suitable for a given application a dye not only needs to have certain chromophoric properties but has also to interact with the matrix in a certain manner. One example is the hematoxylin-eosin stain in histology. This staining method takes advantage of the different staining properties of hematoxylin and eosin towards cellular structures. Hematoxylin stains mainly the cell nuclei blue, whereas eosin stains mainly the cytoplasmic proteins red.
Another important property of dyes is that feasible methods must be present to apply the dye to the matrix. An example is the application of vat dyes to fabrics. Vat dyes are a class of water insoluble dyes that can be made soluble by chemical reduction. Fabric is then immersed in a solution of the reduced form, which is then oxidized in the fabric back to the insoluble dye. Thanks to this application method vat dyes are very color fast. The reduced form of a vat dye is a dye precursor that is transformed into the dye upon a given stimulus at a desired location. In such a case the type of stimulus is not of primary concern as long as it does not have any negative side effects on the dyeing process. However, the formation of the dye from the dye precursor through an external stimulus can also be used to specifically detect said external stimulus. In this case a dye precursor functions as an indicator and the type of stimulus transforming the dye precursor into the dye is of primary interest. We will use the term dye precursor as a general term and the term dye indicator for all kinds of dye precursors when the detection of the said stimulus is of primary interest. Transformation of the precursor typically involves the chemical removal or modification of a labile group (“LG”) of the precursor in a process mediated by the experience of said stimulus. Such stimuli typically include temperature, light (photo-labile or photochromic indicators), electric field (electrochromic indicators), pressure (piezoelectric indicators), ion concentration (e.g. pH indicators) and biochemical reactivity (e.g. enzyme indicators).
Fluorogenic enzyme indicators based on resorufin, fluorescein or umbelliferone derivatives are well known in the art. Although umbelliferone indicators are most widely used, they show various disadvantages. For example, umbelliferones are blue fluorescent under UV light. This is often undesirable because most media exhibit themselves a natural blue fluorescent background. Accordingly, an emission shifted to green, yellow or red would offer an advantage over umbelliferone indicators.
Because umbelliferone (pKa 7.8) only shows significant fluorescence in the deprotonated state, reading of assays only works satisfactorily at alkaline pH, thus limiting the use of these indicators.
Moreover, some indicators, especially ester type indicators (e.g. acetate, nonanoate, inositol phosphate) of umbelliferone and also fluorescein and resorufin are not very stable in aqueous solutions, so that most assays have to be carried out in a relatively short time frame. More stable indicators would also allow performing assays that need longer incubation times, such as enrichment broths. Also, in certain media fluorescence detection might be difficult due to turbid or quenching components in the media. A method for separating the signal from the interfering media would clearly improve the use of such assays.
WO 2010/128120, which was published on 11 Nov. 2010, i.e. after the priority date claimed by the present application, discloses 10H-Indolo[1,2-a]indole staining of bacterial colonies, e.g. in Agar, but not in a low-polarity host with a room-temperature dielectric constant ∈≦10.0.
The publication by Crawford L. A. et al., “Isoindolo[2,1-a]indol-6-one—a new pyrolytic synthesis and some unexpected chemical properties”, Organic & Biomolecular Chemistry, Royal Society of Chemistry, GB, Vol 6, (2008 Jan. 1), pp. 2334-2339, discloses another class of compound, namely iso-indoloindoles. Merely Compound 13 is an indoloindole, but it was only detected as minor product or impurity, and no indications whatsoever are given about its color properties or suitability as a dye.